1. Field of the Invention
This invention relates to a vascular endothelial cell growth factor and to the means and methods for its production in therapeutically significant quantities.
2. Description of the Background Art
Considerable research has been devoted to the morphology and physiology of the secretory cells of the anterior pituitary and pars tuberalis. However, until recently, little was known about the function of the follicular or folliculo-stellate cells (FC), a morphologically well characterized population of granular cells. The FC are stellate cells that send cytoplasmic processes between secretory cells.
A method for the culture of homogeneous populations of FC is described by Ferrara et al., Meth. Enz. (ed. Conn, P.M.), Vol. 124, pp. 245-253 (Academic Press, New York, 1986). The growth pattern and expression of the dome formation by FC in culture and their ultrastructure have been elucidated. Ferrara et al., Am J. Physiol., 252: E304-312 (1987). In addition, FC have been characterized as ion transport elements, possibly involved in the regulation of ion composition and osmolarity of the interstitial fluid in the adenohypophysial cell cords. Ferrara and Gospodarowitz, Biochem. Biophys. Res. Comm., 157: 1376-1382 (1988). In addition, FC produce the angiogenic mitogen basic fibroblast growth factor (bFGF). Ferrara et al., Proc. Natl. Acad. Sci., U.S.A., 84: 5773-5777 (1987).
The gene encoding bFGF, disclosed in Abraham et al., EMBO J. 5: 2523-2529 (1986), does not code for a conventional signal peptide, required for the extracellular transport of proteins according to classical secretory pathways. Walter and Blobel, J. Cell. Biol., 91: 557-561 (1981). Neither does the gene coding for acidic fibroblast growth factor (aFGF), disclosed in Jaye et al., Science, 233: 541-544 (1986). Accordingly, the growth factor is not appreciably secreted into the medium [Moscatelli et al., J. Cell Physiol., 129: 273-277 (1986); Klagsburn et al., Proc. Natl. Acad. Sci. USA, 83: 2448-2452 (1986)], and responsive cell types are dependent on exogenous bFGF for optimal proliferation in culture, even though they may contain significant intracellular concentrations of the mitogen. Neufeld et al., Endocrinology, 121: 597-602 (1987); Schweigerer et al., Endocrinology, 120: 796-802 (1987); Schweiger et al., Exp. Eye Res., 46: 71-80 (1988). It has been suggested that bFGF may be incorporated into the basement membrane and be subsequently released in a soluble form only when the matrix is degraded following the action of specific enzymes. Vlodavsky et al., Proc. Natl. Acad, Sci. USA, 84: 2282-2286 (1987). Such a mechanism of release suggests a role for the growth factor mostly or exclusively in events that involve degradation of the basement membrane or cell lysis, such as organ remodeling, wound healing, or neoplasia. Folkman and Klagsbrun, Science, 235: 442-447 (1987).
Moreover, bFGF and aFGF are both potent mitogens for corneal endothelial cells, lens epithelial cells, BHK-21 fibroblasts, adrenal cortex cells, and keratinocytes, as well as vascular endothelial cells. Gospodarowicz et al., Endocrine Reviews, 8: 95-114 (1987); Baird et al., Recent Prog. Horm. Res., 42: 143-186 (1986).
A vascular endothelial cell mitogen was isolated and described by Plouet and Gospodarowicz, The International Symposium on the Development of the Vascular System, Madison, Wis., Apr. 23-26, 1989, entitled "Isolation and Characterization of a New Vascular Cell Mitogen: Vasculotropin." Most recently, a heparin-binding endothelial cell growth factor called vascular endothelial growth factor was identified and purified from media conditioned by bovine pituitary follicular or folliculostellate cells. Ferrara and Henzel, Biophys. Res. Comm., 161: 851 (1989).
There is a need for a growth factor that, in contrast to aFGF and bFGF, is not sequestered inside the cell source but rather secreted, with resultant direct access to target cells. Such a growth factor may play a more dynamic role in the physiological regulation of vascular endothelial cell proliferation, either in the cyclical growth of blood vessels that takes place in organs such as the corpus luteum [Bassett, Am. J. Anat., 73: 251-259 (1943)] or in the tonic maintenance of the differentiated state of the endothelium in the vascular tree.
There is also a need for a growth factor that is specific for vascular endothelial cells, in contrast to aFGF and bFGF, which are active on a very broad spectrum of cells. Such specificity may be useful therapeutically for conditions in which a selective action on the vascular endothelial cells, in the absence of excessive connective tissue proliferation, is desirable, such as diabetic ulcers or traumatic vascular injuries.
Although a vascular endothelial cell growth factor meeting the above needs can be isolated and purified from natural sources for subsequent use, the relatively low concentration of the protein in FC and the high cost, both in terms of effort and expense, of recovering in commercial quantities purified growth factor from FC hinder its broad-scale use.
Accordingly, it is an object of the present invention to isolate DNA encoding a vascular endothelial cell growth factor and to produce commercially useful quantities of the protein from a therapeutically acceptable source.
It is a further object to obtain the vascular endothelial cell growth factor in a form unaccompanied by the glycosylation associated with the corresponding native growth factor.
It is an additional object to prepare amino acid sequence and other variants of the vascular endothelial cell growth factor that do not substantially adversely affect the biological activity of the protein.
It is yet another object to produce a vascular endothelial cell growth factor completely free of other naturally occurring (source) proteins.
These and other objects of the invention will be apparent from the specification as a whole.